Strain Selection for Biodiesel Production - Biotechnological Applications of Microalgae - page 22

the isolation and the subsequent purification of the algae. It has been suggested that
the techniques normally used to isolate algae may severely limit the number of algal
species that can be readily cultured (Castenholz, 1988). The goals of algae isolation
and screening efforts are to identify and maintain promising algal specimens for cul-
tivation and strain development. Because it is premature to decide on the system of
mass cultivation, new strains should be isolated from a wide variety of environments
to provide the largest range of metabolic versatility possible. Algae can be isolated
from a variety of natural aquatic habitats, ranging from freshwater to brackish water,
to marine and hyper-saline environments, to soil (Round, 1984; Mutanda et al.,
2011). Furthermore, large-scale sampling efforts should be coordinated to ensure
the broadest coverage of environments and to avoid duplication of efforts. The selec-
tion of specific locations can be determined by sophisticated site selection crite-
ria through the combined use of dynamic maps, geographic information systems
(GIS) data, and analysis tools. Ecosystems to be sampled could include aquatic
(i.e., oceans, lakes, rivers, streams, ponds, and geothermal springs, which includes
fresh, brackish, hypersaline, and acidic and alkaline environments) and terrestrial
environments in a variety of geographical locations to maximize genetic diversity.
Collection sites can include public lands as well as various sites within national and
state park systems. In all cases, questions of ownership of isolated strains should
be considered. Additionally, within an aqueous habitat, algae are typically found in
planktonic (free floating) and benthic (attached) environments. Planktonic algae may
be used in suspended mass cultures, whereas benthic algae may find application in
biofilm-based production facilities. Sampling strategies should not only account for
spatial distribution, but also for the temporal succession brought about by seasonal
variations of algae in their habitats (Mutanda et al., 2011).
3.3 ISOLATION TECHNIQUES
For the isolation of new strains from natural habitats, traditional cultivation tech-
niques may be used, such as enrichment cultures (Andersen and Kawachi, 2005).
A preferred preliminary step toward single-cell isolations would be enrichment
cultures with growth media, soil and/or water extract, or supplementing with nutri-
ents such as nitrate, ammonium, and phosphate or a trace metal. Alternatively,
the proximate nutrient composition of the source samples may be analyzed and
supplemented to the growth media. Algae survive under natural environments
despite the fact that natural samples are often deficient in one or more nutrients.
This may be due to the fact that bacterial action, grazing, and death of organ-
isms recycle those nutrients. Sampling reduces the population of specific spe-
cies that help recycle nutrients, and this nutrient stress leads to a decline of the
target species. Enrichment can also be disadvantageous if the target species is
unable to compete with the other autochthonous flora. Hence, selective culturing
is a unique tool suited for enrichment culturing of lipid-producing microalgae.
Once enriched, suspensions containing algae from samples may be centrifuged to
increase the biomass concentration of desired cell density; then diluted in sterile
water and passed through a 60-µm plankton net to remove zooplankton, and col-
lected again using a 0.45-µm glass filter. The cells on the filter should be rinsed